Herein, we report a novel two-step
method for the covalent, site-directed,
and efficient immobilization of proteins on lab-made paper sheets.
First, paper fibers were modified with a peptidic anchor comprising
enzyme recognition motifs. Four different conjugation strategies for
peptide immobilization were evaluated with respect to reproducibility
and fiber loading efficiency. After manufacturing of the peptide-preconditioned
paper, oriented conjugation of the model protein tGFP containing a
C-terminal recognition sequence for either sortase A or microbial
transglutaminase was assessed semiquantitatively by fluorescence measurement
and inspected by confocal laser scanning microscopy (CLSM). The two
enzymes utilized for protein conjugation used the same oligoglycine
peptide anchor, and both proved to be suitable for controlled oriented
linkage of substrate proteins at physiological conditions.
Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α‐carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in‐depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA‐mediated formation of thioether bridges, which were identified mass‐spectrometrically. In a proof‐of‐principle study, we re‐engineered subtilosin A to a thioether‐bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides.
Sactipeptide sind ribosomal synthetisierte Peptide, die eine einzigartige Verknüpfung von Schwefel und α-Kohlenstoffen enthalten. Die Bildung von Thioetherbrücken wird in diesen Molekülen durch Sactisynthasen katalysiert. Diese spezielle Art der Verknüpfung verleiht Sactipeptiden eine erhöhte strukturelle, thermische und proteolytische Stabilität, was sie zu attraktiven Gerüsten für die Entwicklung neuer Biotherapeutika macht. In diesem Artikel berichten wir über eine Studie zur Substrattoleranz der Sactisynthase AlbA, die die Bildung von Thioetherbrücken im Sactipeptid Subtilosin A katalysiert. Wir haben eine Modifikationsstelle innerhalb dieses Sactipeptids identifiziert, die ein Peptid-Engineering ohne Beeinträchtigung der Bildung von Thioetherbrücken ermöglicht. Eine Reihe von natürlichen und hybriden Sactipeptidkonstrukten wurde hergestellt, um die AlbA vermittelte Bildung von Thioetherbrücken zu untersuchen und diese massenspektrometrisch zu identifizieren. In einer Proofof-Principle-Studie haben wir Subtilosin A mit einer neue Funktion ausgestattet, ein Thioether-verbrücktes Streptavidin-bindendes Peptid generiert und damit die Tür für das funktionelle Engineering von Sactipeptiden weiter geöffnet.
Due to the large evolutionary distance between birds (Aves) und humans, immunization of chickens with human proteins results in a strong response of the bird’s adaptive immune system to proteins of mammalian origin. Additionally, chicken-derived antibodies display less undesired cross-reactivity in analytical setups than conventional rodent-derived antibodies. Due to these features as well as the facile amplification of antibody-coding genes, chicken-derived antibodies emerged as promising molecules for the immunotherapy and various biotechnological applications.
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